Weapons of mass destruction are, unfortunately, a fact of life as we approach the 21st Century. Of grave concern are bacterial agents, such as Bacillus anthracis and Clostridium botulinum, that could be used by rogue or aggressor countries and/or international terrorists. Such biological agents could be easily dispersed in urban and/or battlefield areas using a multitude of delivery devices. Just the threat of such an attack can cause considerable harm to the threatened population or area as well as national security. Moreover, the risk of such threats and attacks will likely increase as it becomes increasingly easy to prepare, use, and/or deliver such biological agents anywhere in the world. Thus, highly sensitive and selective detection methods are vital to our national security both at home and abroad.
The detection methods currently available generally lack the desired sensitivity, selectivity, and/or speed for such bacterial agents as Bacillus anthracis and Clostridium botulinum. Currently available detection methods include, for example, PCR with fluorescence detection, fluorescent antibody staining techniques (FAST), partichrome analyzers, bioluminescence-based systems, electrochemiluminescence-based systems, and stimulated induced fluorescence techniques for on-site and remote detection. Some of these techniques (e.g., PCR with fluorescence detection, ELISA, and FAST), although having high specificity, have relatively low sensitivity (minimum of 104 to 106 spores for detection) and are time consuming (generally requiring 0.5 to 6 hours for results). See, e.g., Redkar et al., “Rapid Detection of Select Pathogenic Bacteria by Real Time PCR,” Abstract, Scientific Conference on Chemical and Biological Defense Research, Aberdeen Proving Ground (1998); Ibrahim, “Detection of Biological Agents Using Probe-Based PCR Assay,” Abstract, Scientific. Conference on Chemical and Biological Defense Research, Aberdeen Proving Ground (1998); Gatto-Menking et al., “Rapid Post PCR Protection Using IGEN's Origen Analyzer,” Abstract, Scientific Conference on Chemical and Biological Defense Research, Aberdeen Proving Ground (1998); Bruno et al., “Development of Selex DNA Aptamers for the Detection of Anthrax Spores,” Abstract, Scientific Conference on Chemical and Biological Defense Research, Aberdeen Proving Ground (1998); and Nagata et al., “Development of Enzyme-linked Immunosorbent Assay (ELISA) to Anthrax for the Persian Gulf,” Defense Information System Agency, ADA297350 (1995). Bioluminescence- and electrochemiluminescence-based systems, although having increased sensitivity (minimum of about 200 spores for detection) and being somewhat faster, suffer because of reduced specificity. See, e.g., Gatto-Menking et al., “Rapid Post PCR Protection Using IGEN's Origen Analyzer,” Abstract, Scientific Conference on Chemical and Biological Defense Research, Aberdeen Proving Ground (1998); and Bartoszcze et al., “The Sensitivity of the Bioluminescence Method Regarding Microbiological Detection in Contaminated Water and on Surfaces,” Abstract, Scientific Conference on Chemical and Biological Defense Research, Aberdeen Proving Ground (1998). Additionally, stimulated induced fluorescence methods have be used to detect or target the amino acid tryptophan. However, since tryptophan is common to most biological materials, techniques relying on its detection have very poor selectivity.
There remains, therefore, a need for simple detection methods and devices for bacterial agents such agents as Bacillus anthracis and Clostridium botulinum. Moreover, there remains a need for such detection methods and devices having high sensitivity and high selectivity and which can provide real-time data. There remains a need for such detection methods and devices which can be carried by personnel who may be exposed to such bacterial agents and provide warning so that protective measures can be taken to avoid or minimize exposure to such bacterial agents. There also remains a need for such detection methods and devices which have low rates of false positives. This invention provides such methods and devices for the detection of bacterial agents such as Bacillus anthracis and Clostridium botulinum. This invention also provides such methods and devices for the detection of viable spores of Bacillus anthracis and Clostridium botulinum. Moreover, these and other advantages and benefits of the present methods and devices will be apparent from a consideration of the present specification.